Influenza virus RNA segment 8 codes for two distinct proteins, NS1 and NS2, that are translated from separate mRNA's. Mapping and sequencing studies have shown that the NS1 mRNA is a colinear transcript and NS2 mRNA contains a spliced region. In order to investigate the splicing potential of influenza virus mRNA derived from virion RNA segment 8, cloned full-length NS DNA was inserted into the late region of an SV40 expression vector and the recombinant used for infection of primate cells. Sizing by nuclease S1 analysis and nucleotide sequencing indicated that both interrupted and uninterrupted mRNA's containing influenza NS sequences were synthesized in cells infected with the recombinant. The sequences found at the junction of the interrupted mRNA were identical to those found in the NS2 mRNA produced in influenza virus infected cells. These studies establish that during influenza virus infection processing of the NS mRNA transcript involves a mechanism of splicing similar to that which occurs with DNA-directed RNA transcription. Our observations thus eliminate other possible explanations for interrupted mRNAs such as "transcription from defective interfering particles and transcriptional jumping."